Intro to BioHacking

8/12/2019 Intro to BioHacking

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INTRO TOBIOHACKINGOr How I learned to stop worrying and lovethe zomie apo!alypse"


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#tr$!t$re

%hat is Bioha!&ing' Crash !o$rse in (ole!$lar Biology Key Con!epts and Tools )or a io la

%hat are other ioha!&ers $p to' %hat prolems are the !omm$nity )a!ing*

how !an we solve them'

+is!$ssion , o$r pro-e!t ideas and goals


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B$t .irst/

A dis!laimer000

(e+r Roert Neville


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#o000 Bioha!&ing* %T.'

Two main streams o) development 1resear!h/

Biologi!al resear!h 1 engineering Cloning

2random3 m$tagenesis

4hysi!al 1 !hemi!al resear!h and engineering B$ilding open5so$r!e* !ost5e))e!tive la e6$ipment


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Central +ogma" o) mol iol

In)ormation is en!oded in o$r +NA

O$r +NA !ontains )o$r ases/ A C T G

These se6$en!es are re!ipes" )orman$)a!t$ring proteins* whi!h do all the wor& inthe !ell

H$mans have 78 illion ase pairs en!oding

appro9 :;5:


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+NA

RNA

4rotein

%or&

Transcription:A !omple9 o) proteinsre!ognise a promoterse6$en!e" at the start o)a gene

This protein !omple9re!r$its RNA polymerase

RNA polymerase $ses+NA as a template to

ma&e a !omplementarystrand o) RNA

RNA )loats away )orpro!essing000

+NA is le)t $n!hanged


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mRNA is a template )or proteinman$)a!t$re

mRNA )eeds thro$gh a ribosome*whi!h re!r$its protein s$$nits

#$$nits get assemled into peptide!hain* $ntil end o) the mRNA isrea!hed

+NA

RNA

4rotein

%or&


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Central +ogma" 5 re!ap

+NA , !entral in)ormation store...transcribed to...RNA , wor&ing !opy* a!ts as a template...translated to...4rotein , the !ompleted ma!hine

+NA

RNA

4rotein

%or&


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Central +ogma" , >r000 sorto)000 All the aove is !orre!t000 $t very in!omplete=

+NA se6$en!es !an e str$!t$ral* reg$latory or!oding 2or any !omination o) these=3

RNA se6$en!es !an e str$!t$ral* reg$latory*enzymati!ally a!tive* !oding 2or !ominations o)

these=3(eta5in)ormation/

#pli!e variants?o!alisation signals(odi)i!ation signals

000et!* et!000

However* this is good eno$gh )or most !loningwor&=

+NA

RNA

4rotein

%or&


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Introd$!tion to !loning

Cloning is 2$s$ally3 NOT ao$tma&ing !opies o) whole

organisms=

I &now* I was disappointed too000


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The aim o) !loning is to end $p with aplasmid that we !an p$t into o$r targetorganism* where it will e))i!iently e9press

o$r desired gene0 @ses in!l$de/

protein prod$!tion 2e0g0 Ins$lin man$)a!t$re3

improved s$rvival 2e0g0 Antiioti! resistan!e3

Red$!ed s$rvival 2new v$lnerailities to dr$gs3 modi)ied ehavio$r 2new rea!tions to stim$li3

Creating )$sion proteins

000et!


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>le!trophoresis allows $s toseparate* vis$alise ande9tra!t desired )ragments*ready to p$t the right onestogether000


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Trans)e!t 1 Trans)orm 1 Transd$!e plasmid into yo$r !ells

Then need to sele!t )or !ells that have s$!!ess)$lly ta&en $p thegene

(ost !ommon approa!h is antiioti! sele!tion

>nd res$lt/ a p$re !$lt$re o) easties e9pressing the novel

!omination o) gene2s3 yo$ve !hosen* in the !onditions yo$vedesigned


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Core te!hni6$es

Polymerase Chain ReactionA method to rapidly !reate many !opies o) a +NA se6$en!e

essential )or modern mole!$lar iology

Two primer" se6$en!es aremi9ed with the template +NA* a+NA polymerase* and dNT4s0

The temperat$re is !y!led toprogress the rea!tion ea!h

!omplete !y!le do$les then$mer o) !opies present

Res$lting +NA !an e $sed )ordete!tion* 6$anti)i!ation* !loning*m$tagenesis* et!000


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Core te!hni6$es

Variants on the PCR:

RT54CR to dete!t RNA strands

64CR 2or RT564CR3 to !o$nt n$mer o) strands

($tageni! 4CR , to !reate da$ghter" strands in!l$ding a newm$tation


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Core te!hni6$es

Western blotting and E!S"

Antiodies ind to andre!ognise spe!i)i! proteins

Can e $sed to immoilise yo$rproteins or -$st to dete!t them

(a&e yo$r own 2hard* time!ons$ming* $nethi!al and veryillegal=3 or !ommer!iallyavailale


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Core te!hni6$es

Western blotting and E!S"

These te!hni6$es $sed to!on)irm or meas$re

e9pression o) yo$r protein

>le!trophoresis is $sed toseparate proteins y size

Then protein smear" is

proed with antiody todete!t yo$r spe!i)i! targetsho$ld all e at the sameheight on the gel

>?I#A is similar* $t no

ele!trophoresis000


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Key tools D reso$r!es )or aio la

>nvironment/ !leanliness* !ontainment*

disposal Reagent handling/ need to meas$re tiny

vol$mes 1 masses

Centri)$ge

Thermal !y!ler

>le!trophoresis tan& 2D power s$pply3

In!$ator D water ath


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PipettesHandling a wide range o)

vol$mes* some in sterile!onditions

l )


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Key tools D reso$r!es )or aio laCentri&'geNeed to spin samples )or separating

layers o) !hemi!als* pelleting +NA)or p$ri)i!ation* $sing vario$s la&its000 An essential tool0

#ensile minim$m is to sa)ely spin a :gram sample at *;;;g

- A!hievale with a dremel)$ge"* i)yo$ get someone else to hold it 3

?arger masses are desirale 2e0g0


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K l D )


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!nc'bator ( Waterbath

Eario$s organisms strongly pre)erparti!$lar temperat$res and sho$ld egrown in a )airly narrow temperat$re

range0

As a !onse6$en!e* many enzymes 2esprestri!tion enzymes and polymerases3are very ine))i!ient o$tside narrowtemp ranges0

There)ore* need somewhere warm5$t5not5hot to grow $gs and r$nrea!tions=

2(ost $gs and enzymes in reg$lar $seare est at 8F0


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Thermal cycler

>ssential )or 4CR

Basi!ally -$st a programmale

hotplate a typi!al programme is/

#TART/


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K t l D )


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Key tools D reso$r!es )or aio laReagentsA lot o) mole!$lar iology !an e done with &its* whi!h streamline pro!esses

and avoid nasty !hemi!als000 >9pensive

A lot o) wor& going into avoiding nastier and more e9pensive !hemi!als within

the !omm$nity

Care)$l planning 2e0g0 Biori!&s3 !an minimise the ne!essary reagent lirary

Restri!tion enzymes are a ig !hallenge

The !omm$nity needs to settle on model organisms to wor& with000 Eersatilityand sa)ety are !on!erns=


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Intro to BioHacking

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